Sickle cell trait individuals usually have about 60% HbA and 40% HbS. Those who also have an alpha-thalassemia gene(s), which decreases production of alpha chains, have an even lower proportion (25 to 35%) of HbS. To provide a model for the effect of the alpha-thalassemia gene(s) on HbA and HbS levels, we measured the amounts of the two hemoglobins that were reconstituted from various mixtures of native alpha, beta A, and beta S chains. Various limiting amounts of heme-containing 3H-labeled alpha chains were added to replicate mixtures containing beta S and beta A chains present in proporations (40% beta S, 60% beta A) similar to those of Hbs S and A in the blood of sickle trait individuals without alpha-thalassemia gene(s). After incubation at 4 degrees C for 60 min., the relative amounts of 3H-radioactivity as HbS and HbA were determined by electrophoresis on cellulose acetate strips. The HbS formed was less than 40% of the two product hemoglobins at all levels of 3H-alpha chains used, i.e., 0 less than alpha/total beta ration less than 1. However, when the ratio of alpha to total beta chains was similar to that of newly synthesized chains in the reticulocytes of sickle trait individuals with an alpha-thalassemia gene(s) (alpha/total beta synthesis ratio equals 0.65 to 0.80), the relative amounts of HbS formed in the cell-free system were greater than those found in vivo. Incubation at 37 degrees C and addition of KCl and 2,3-diphosphoglycerate at concentrations similar to those inside human red cells also did not produce the relative deficit of HbS observed in vivo. We have shown previously that less HbS than HbA was formed in these cell-free reactions because of a lower affinity of alpha chains for beta S chains than for beta A chains. Our recent results suggest that other factors such as a rapid turnover of newly synthesized beta S chains, in addition to a difference in subunit chain affinities, probably contribute to the enhanced deficit of HbS in sickle trait individuals with an alpha-thalassemia gene(s).